Project Summary As knowledge of the molecular drivers of oncogenesis and tumor progression has grown, so too has our ability to deploy more effective and less toxic molecular therapies. For example, targeted cancer therapies such as ALK and EGFR inhibitors in lung cancer are leading to improved clinical outcomes. However, not all patients benefit from this emerging precision medicine approach, such as patients with KRAS-mutant cancers, and those patients who do benefit initially from targeted therapy ultimately succumb to tumor progression due to drug resistance. Gaining a better understanding of the aberrant cell signaling regulation driving cancer initiation, progression, and drug resistance is essential to expand, and improve, molecular treatment options for patients to extend their survival. A major gap in the field is that very little is known about the potential presence and function of subcellular structures that can organize cell signaling in a cancer-specific manner to promote cancer pathogenesis. By studying the molecular determinants of response and resistance to ALK targeted therapy in ALK gene rearrangement lung adenocarcinoma, we discovered that oncogenic ALK gene rearrangements are uniquely and exquisitely dependent on RAS-RAF-MEK-ERK (RAS/MAPK) signaling for growth and survival. Our studies revealed that the basis of the dependence is that this oncogenic ALK activates RAS from an intracellular, cytoplasmic compartment instead of a lipid-membrane compartment in cells. This was surprising because receptor kinases such as native ALK and RAS both canonically signal exclusively from a lipid-membrane compartment such as the plasma membrane. Our findings prompt the intriguing hypothesis that RAS signaling can occur from a protein granule in the cytoplasm, rather than a lipid- membrane compartment in certain cancers. We propose four Specific Aims that leverage genetic, proteomic, biophysical, and cell biological studies to test this hypothesis, with the goal of demonstrating for the first time in mammalian cells that RAS signaling can emanate from the cytoplasm, in an organized protein-based structure that lacks lipid-membranes. We will test this hypothesis initially in lung cancers with oncogenic ALK and expand to those with oncogenic signaling caused by other aberrant kinase gene fusions that may signal from a similar intracellular protein-based platform. If our hypothesis is true, the findings will transform our understanding of the molecular basis of cancer and overturn 25 years of dogma that holds that RAS signaling can only occur from a lipid-membrane compartment. The findings will generate a new understanding of the role of protein granules in cancer pathogenesis, thereby ascribing an unanticipated biological function for this emerging class of subcellular structures. Our efforts hold important implications for designing entirely novel diagnostic and therapeutic strategies to exploit the pathognomonic subcellular organization of oncogenic signaling to improve treatment options for patients in the future. This project could have broad impacts on the understanding of cancer pathogenesis and pave the way for new molecular strategies to better control cancer.